PROJECTSUMMARY/ABSTRACT Autosomaldominantmutationsinvacuolarproteinsorting35(VPS35)havebeenidentifiedasacausalPD gene,playingaroleinthedevelopmentoflate-onsetPD.VPS35functionsasascaffoldingproteinforretromer complexthatmediatesrecyclingofcargoproteins(transmembraneproteins)fromendosomestothetrans- Golgiapparatusortheplasmamembrane.Therefore,mutationsinVPS35couldpreventorlimititsdeliveryof thecargoes,whicharecrucialforthesurvivalofdopaminergicneurons.Hence,adisruptionintherecycling pathwayofthesecargoesmayplayacrucialroleinthedemiseofdopaminergicneuronsinthesubstantia nigraofpatientswithVPS35mutations.AgreaterunderstandingofbiologyofVPS35andthepathophysiology ofmutantVPS35areessentialtothedevelopmentoftherapeuticinterventionsaimedatpreventingtheonset and/orretardingprogressionofPD.Nonetheless,thefunctionalconsequencesoftheVPS35geneticvariations inPDhavenotyetbeendiscovered.TobetterunderstandthepathogenicinvolvementofVPS35mutationsin vivo,wegeneratedatetracyclineconditionalhumanVPS35transgenic(Tg)mousewhereexpressionof mutanthumanD620NVSP35orwild-type(WT)humanVPS35proteinsisachievedinthenigrostriatal dopaminergicpathway,underthecontrolofthedopaminepathway-specifictyrosinehydroxylase(TH)-tTA driver.Utilizingthismousemodel,inaim1,wewillstudyneurochemical,neuroanatomicalandbehavioral changesusinghigh-performanceliquidchromatography,unbiasedstereologicaltechniques,andbehavioral testinginthesemiceastheyage.Inparticular,wewillexplorewhethertheoverexpressionofmutantD620N VPS35indopaminergic(DA)neuronsmayinducelossofdopaminergicneuronsduringaging.Importantly,our preliminarystudyindicatesthattherearerobustandprogressivedegenerationinthesubstantianigraofthe TH-tTA/TetP-D620NVPS35mice.Inaddition,thereisanintriguingbutpoorunderstandingofthepathogenic interplaybetweentheVPS35mutationandmitochondriadysfunctioninPD.Ourpreliminaryresultindicates thatVPS35interactswithKeap1andtheinteractionbetweenKeap1andD620NVPS35leadstoaccumulation ofKeap1,akeyregulatorofNrf2,andaconcomitantdecreaseinproteinlevelsandactivityofNrf2,amaster regulatorofoxidativestress.Inaddition,thedysregulationofKeap1/Nrf2levelsmediateD620NVPS35- inducedDAneuronaltoxicityandmitochondriadysfunctioninhumanDAneurons.Inaim2,wewill characterizeapotentialdefectinmitochondrialqualitycontrolintheD620NVPS35Tgmicerepresenting degenerationofDAneuronsandtheroleofthederegulationofKeap1/Nrf2levelsinregulatingthesedefects. Moreover,wewilldeterminewhethersuppressionofKeap1accumulationrescuesthelossofDAneuronsand mitochondriadysfunctionsinD620NVPS35Tgmice.Thisproposalmayprovideaneworvaluablegenetic mousemodelforPDwithdopaminergicneurodegenerationandanewinsightofVPS35retromerfunctionin lossofdopaminergicneuronsandmitochondriadysfunction.